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Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is effective in patients with advanced B-cell acute lymphoblastic leukemia (B-ALL). However, efficacy data is sparse in subgroups of patients with high-risk features such as BCR-ABL+, TP53 mutation, extramedullary disease (including central nervous system leukemia) or posttransplant relapse. It is also uncertain whether there is an added benefit of transplantation after anti-CD19 CAR T-cell therapy. We conducted a phase 1/2 study of 115 enrolled patients with CD19+ B-ALL. A total of 110 patients were successfully infused with anti-CD19 CAR T cells. In all, 93% of patients achieved a morphologic complete remission, and 87% became negative for minimal residual disease. Efficacy was seen across all subgroups. One-year leukemia-free survival (LFS) was 58%, and 1-year overall survival (OS) was 64% for the 110 patients. Seventy-five nonrandomly selected patients (73.5%) subsequently received an allogeneic hematopoietic stem cell transplant (allo-HSCT). LFS (76.9% vs 11.6%; P < .0001; 95% confidence interval [CI], 11.6-108.4) and OS (79.1% vs 32.0%; P < .0001; 95% CI, 0.02-0.22) were significantly better among patients who subsequently received allo-HSCT compared with those receiving CAR T-cell therapy alone. This was confirmed in multivariable analyses (hazard ratio, 16.546; 95% CI, 5.499-49.786). Another variate that correlated with worse outcomes was TP53 mutation (hazard ratio, 0.235; 95% CI, 0.089-0.619). There were no differences in complete remission rate, OS, or LFS between groups of patients age 2 to 14 years or age older than 14 years. Most patients had only mild cytokine release syndrome and neurotoxicity. Our data indicate that anti-CD19 CAR T-cell therapy is safe and effective in all B-ALL subgroups that have high-risk features. The benefit of a subsequent allo-HSCT requires confirmation because of nonrandom allocation. This trial was registered at www.clinicaltrials.gov as #NCT03173417.

Backgrounds As CAR T-cell therapy is a highly personalized therapy, process of generating autologous CAR-T cells for each patient is complex and can still be problematic, particularly for heavily pre-treated patients and patients with significant leukemia burden. Here, we analyzed the feasibility and efficacy in 37 patients with refractory/relapsed (R/R) B-ALL who received CAR T-cells derived from related donors. Patients and Methods From April 2017 to May 2020, 37 R/R B-ALL patients with a median age of 19 years (3-61 years), were treated with second-generation CD19 CAR-T cells derived from donors. The data was aggregated from three clinical trials (www.clinicaltrials.gov NCT03173417; NCT02546739; and www.chictr.org.cn ChiCTR-ONC-17012829). Of the 37 patients, 28 were relapsed following allogenic hematopoietic stem cell transplant (allo-HSCT) and whose lymphocytes were collected from their transplant donors (3 HLA matched sibling and 25 haploidentical). For the remaining 9 patients without prior transplant, the lymphocytes were collected from HLA identical sibling donors (n=5) or haploidentical donors (n=4) because CAR-T cells manufacture from patient samples either failed (n=5) or blasts in peripheral blood were too high (>40%) to collect quality T-cells. The median CAR-T cell dose infused was 3×105/kg (1-30×105/kg). Results For the 28 patients who relapsed after prior allo-HSCT, 27 (96.4%) achieved CR within 30 days post CAR T-cell infusion, of which 25 (89.3%) were minimal residual disease (MRD) negative. Within one month following CAR T-cell therapy, graft-versus-host disease (GVHD) occurred in 3 patients including 1 with rash and 2 with diarrhea. A total of 19 of the 28 (67.9%) patients had cytokine release syndrome (CRS), including two patients (7.1%) with Grade 3-4 CRS. Four patients had CAR T-cell related neurotoxicity including 3 with Grade 3-4 events. With a medium follow up of 103 days (1-669days), the median overall survival (OS) was 169 days (1-668 days), and the median leukemia-free survival (LFS) was 158 days (1-438 days). After CAR T-cell therapy, 15 patients bridged into a second allo-HSCT and one of 15 patients (6.7%) relapsed following transplant, and two died from infection. There were 11 patients that did not receive a second transplantation, of which three patients (27.3%) relapsed, and four parents died (one due to relapse, one from arrhythmia and two from GVHD/infection). Two patients were lost to follow-up. The remaining nine patients had no prior transplantation. At the time of T-cell collection, the median bone marrow blasts were 90% (range: 18.5%-98.5%), and the median peripheral blood blasts were 10% (range: 0-70%). CR rate within 30 days post CAR-T was 44.4% (4/9 cases). Six patients developed CRS, including four with Grade 3 CRS. Only one patient had Grade 3 neurotoxicity. No GVHD occurred following CAR T-cell therapy. Among the nine patients, five were treated with CAR T-cells derived from HLA-identical sibling donors and three of those five patients achieved CR. One patient who achieved a CR died from disseminated intravascular coagulation (DIC) on day 16. Two patients who achieved a CR bridged into allo-HSCT, including one patient who relapsed and died. One of two patients who did not response to CAR T-cell therapy died from leukemia. Four of the nine patients were treated with CAR T-cells derived from haploidentical related donors. One of the four cases achieved a CR but died from infection on day 90. The other three patients who had no response to CAR T-cell therapy died from disease progression within 3 months (7-90 days). Altogether, seven of the nine patients died with a median time of 19 days (7-505 days). Conclusions We find that manufacturing CD19+ CAR-T cells derived from donors is feasible. For patients who relapse following allo-HSCT, the transplant donor derived CAR-T cells are safe and effective with a CR rate as high as 96.4%. If a patient did not have GVHD prior to CAR T-cell therapy, the incidence of GVHD following CAR T-cell was low. Among patients without a history of transplantation, an inability to collect autologous lymphocytes signaled that the patient's condition had already reached a very advanced stage. However, CAR T-cells derived from HLA identical siblings can still be considered in our experience, no GVHD occurred in these patients. But the efficacy of CAR T-cells from haploidentical donors was very poor. Disclosures No relevant conflicts of interest to declare.

The high incidence of tuberculosis (TB) has brought serious social burdens and it is urgent to explore the mechanism of TB development. This study was conducted to analyze the role of lncRNA-miRNA-mRNA regulatory network and its contained nodes involved in TB to identify crucial biomarkers for early diagnosis of TB. Long-noncoding RNAs (lncRNAs), messenger RNA (mRNAs) and microRNAs (miRNAs) expression profiles of TB patients and healthy individuals were downloaded from the GSE34608 dataset. Weighted gene co-expression network analysis (WGCNA) was performed to identified the key modules related to TB and the highly related mRNA-lncRNA pair in the module. Based on highly related mRNAs and lncRNAs in greenyellow module, lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was constructed. The DE-mRNAs in the network were functionally enriched with Gene ontology (GO) and Gene set enrichment analysis (GSEA). Least absolute shrinkage and selection operator (LASSO) algorithm and receiver operating characteristic curve (ROC) were used to construct and evaluate the prediction model of TB. We identified 3267 DE-mRNAs, 484 DE-lncRNAs and 69 DE-miRNAs between the TB and healthy subjects, from which 8 DE-mRNAs, 14 DE-lncRNAs and 3 DE-miRNAs were used to construct the ceRNA network. The genes contained in the ceRNA network were mainly enriched in neutrophil mediated immune response, including neutrophil activation, degradation and signal transduction. ROC analysis revealed that has-miR-140–5p, has-miR-142–3p and the LASSO cox prediction model based on HMGA1 and CAPN1 have potential value for forecasting TB (AUC > 0.7). Hence, our study provides a new perspective from the lncRNA-miRNA-mRNA ceRNA regulatory network for TB diagnosis and treatment.

Introduction Chimeric antigen receptor (CAR) T -cell therapy has demonstrated high response rates among patients with B cell malignancies yet remission durability and safety could be improved. We have developed a novel double-chain chimeric receptor Synthetic T Cell Receptor and Antigen Receptor (STAR) consisting of 2 protein modules each containing an antibody light or heavy chain variable region, the T Cell Receptor (TCR) a or b chain constant region fused to the OX-40 co-stimulatory domain, with the 2 modules linked by a self-cleaving Furin-p2A sequence that allows the modules to be proteolytically separated and reconstituted (Fig. 1A). Here, we report pre-clinical and first-in-human phase I trial results of CD19 STAR-T cell therapy for CD19+ R/R B-ALL. Methods Peripheral blood (PB) mononuclear cells were obtained from healthy donors and patients for the pre-clinical and clinical studies, respectively. T-cells were transduced with the STAR lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR T-cell antitumor function. For the clinical trial, from Dec. 2019 to Jun. 2020, 18 CD19+ R/R B-ALL patients (M/F 10:8) with a median age of 22.5 years (range: 6-68) were enrolled (NCT03953599). Patients received a conditioning regimen of IV fludarabine (25mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single STAR T-cell infusion. Once patients achieved complete remission (CR), they were given the option to proceed to consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) or not. Results In preclinical studies, we found CD19 STAR T-cells to be superior to conventional CAR (BBz CAR) measured by the following parameters: 1) faster/stronger T-cell activation within 3 hours (76.67±2.621% vs 46.4±9.318%; p=0.0253); 2) higher cytokine production (4100.92±174.4 pg/ml vs 2556.78±563.39 pg/ml; p1:1, p In the phase I trial, the median observation time was 69 (20-180) days. The median pre-treatment bone marrow (BM) blast level was 7.0% (0.1%-86.6%). All 18 patients received a single infusion of STAR T-cells at a median dose of 1×106/kg (5×105/kg-2.5×106/kg): low dose (5×105/kg) (n=3), medium dose (1×106/kg) (n=8) and high-dose (2-2.5×106/kg) (n=7). Three early enrollees subsequently received a second consolidation infusion of STAR T-cells at 1×106/kg (n=2) and 2×106/kg (n=1). The median STAR T-cell production time was 9 (7-13) days with a transduction efficacy of 57.4% (41.0%-78.2%). Two weeks post STAR T-cell infusion, 18/18 (100%) patients achieved CR with a negative minimal residual disease (MRD) status. After a median of 57 (43-66) days following STAR T-cell therapy, 8/18 patients made a choice to pursue consolidation allo-HSCT and all have remained in CR after a median follow-up of 110 (75-180) days. Of the 10 patients who did not undergo allo-HSCT, 1 relapsed on day 58 and died from relapse on day 63. This patient had a pre-CAR T-cell BM blast level of 86.6% with central nervous system leukemia. Another patient became MRD-positive with 0.09% blasts on day 30 per flow cytometry (FCM). The other 8 patients have remained in CR. Despite the achievement of a high CR rate, cytokine release syndrome (CRS) occurred only in 10/18 (55.6%) patients with 8 Grade I, and 2 Grade II CRS. Two patients developed Grade III neurotoxicity. After STAR T-cell infusion, CD19 STAR T-cells in PB were followed by qPCR and FCM. We saw high in vivo proliferation and persistence regardless of the infusion dose. The median peak level was reached on day 8.5 (day 4-10) with 4.9×104 (0.104-175×104) copy number/ug PB genomic DNA detectable at 6 months. Conclusion This study demonstrates the superiority of STAR T-cells compared to conventional CAR T-cells in terms of signaling capacity, cytokine production capability and anti-tumor potency in an animal model. The Phase I first-in-human study demonstrated technical feasibility, clinical safety and efficacy of STAR-T in treating CD19+ R/R B-ALL. A high CR could be achieved on day 14 with low toxicity. Longer-term observation of these patients and studies of larger patient cohorts are warranted. Disclosures No relevant conflicts of interest to declare.

Introduction Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated promising efficacy in patients with relapsed and refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, relapse after CART remains a major issue. Here, we analyzed the factors related to long-term efficacy, including overall survival (OS), leukemia-free survival (LFS) and cumulative relapse rate (CRR), following CAR-T therapy in 231 R/R B-ALL patients who achieved complete remission (CR) within one month after CAR T-cell therapy. Patients and Methods From April 2017 to March 2019, 254 patients with R/R B-ALL were enrolled onto one of five different clinical trials (NCT03173417; ChiCTR-ONC-17012829; NCT02546739; ChiCTR1800016541; and NCT03671460) at our center and received a second generation CD19+ CAR T-cell infusion. The median infused CAR T-cell dose was 3×105/kg (range: 0.2-10×105/kg). The CAR-T/T-cell ratio and the CD19+ B lymphocyte percentage in PBLC samples from 159 of the patients were analyzed using flow cytometry on day 0, 4, 7, 11, 14, 21, and 30 following CAR T-cell infusion. We performed single continuous variate factors analysis on the influence of the CAR-T/T-cell ratio and the percentage of CD19 + B-lymphocytes in day 30 post-infusion PBLC samples on the OS, LFS, and CRR. We also analyzed the impact of patient age, BM blast count, CAR-T-cell dose, and the interval time between CAR-T-cell therapy and consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) on OS and LFS. Results Among 254 patients, 231 cases achieved CR within one month after CART therapy. A total of 211 CR patients had long-term follow-up of more than 30 days with a median follow-up of 12 months (1 to 29 months). On day 30 post CAR T-cell infusion, the median CAR-T/T-cell ratio in PBLC samples was 0.51% (range: 0%-44.8%), with 59 of 169 patients (34.9%) having a CAR-T/T-cell ratio of ≥1% and 85 of 169 patients (50.3%) with a CAR-T/T-cell ratio of ≥0.5%. The median percentage of CD19+ B lymphocytes in PBLC on day 30 was 0.0% (range: 0.0%-9.4%), of which 157 of 169 patients (92.9%) had Using a single continuous variate factors analysis, we found that increasing BM blasts and percentage of CD19+ B-lymphocytes in PBLC samples on day 30 correlated with a worse OS and LFS (Table 1). BM blasts of ≥70% were statistically significantly correlated with a worse OS and LFS when compared to BM blasts of The remaining 184 patients in CR received a consolidation allo-HSCT after a median interval time of 67 days post CAR T-cell therapy (range: 30-334 days). Thirty-two of these patients (17%) relapsed with a median time to relapse of 221 days (57-490 days). The remaining 27 patients received CAR T-cell therapy only and 11 (41%) relapsed with a median time to relapse of 100 days (53-398 days). None of the four factors above had an influence on the CRR in either the bridging into allo-HSCT group or the CAR-T only group (Table 2). Conclusions Using a single continuous variate factors analysis, we found that a high BM blast count and the percentage of CD19+ B-lymphocytes in PBLC samples from R/R ALL patients on day 30 predicted a worse OS and LFS while age, the CAR-T/T-cell ratio on day 30, CAR-T cell dose, and the interval time between CAR-T cell infusion and allo-HSCT had no clear impact on long-term outcomes. Disclosures No relevant conflicts of interest to declare.

Aim: To assess the efficacy of dendritic cells-cytokine induced killer (DC-CIK) and natural killer (NK) cell-based immunotherapy in treating the low- and intermediate-risk acute myeloid leukemia. Patients & methods: DC-CIK or NK cells were infused once every 3 months for 2–4 cycles to 85 patients. Results & conclusion: The 5-year overall survival (OS) and relapse-free survival (RFS) rates were 90.5 and 65.2%, respectively. The OS of the very favorable, the favorable and the intermediate-risk groups was 94.4, 86.3 and 93.3% (p = 0.88), and the RFS 83.3, 81.8 and 62.2% (p = 0.14), respectively. The OS and RFS of the 60 patients treated with DC-CIK alternating with NK cells were better than the 25 patients treated with DC-CIK or NK alone (96.5 vs 71.2%; p = 0.003. 79.5 vs 28.9%; p < 0.001).

Introduction Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has demonstrated high success for B-cell acute lymphoblastic leukemia (B-ALL). Despite the initial high complete remission (CR) rate, about half of patients (pts) relapse at 1 year. CD19 antigen loss was observed in a significant number of relapsed patients. CD22 is another leukemic marker that often expressed on the surface of CD19- relapsed B-ALL blasts. We have developed a bispecific CAR construct targeting CD19 and CD22. Here we report results from a phase Ⅰ clinical trial of CD19/CD22 (GC022) dual CAR-T to evaluate the safety and feasibility of treating patients with relapsed/refractory B-ALL. Methods The CD19/CD22 dual CAR-T cells were manufactured in a cGMP facility. Patients' peripheral blood (PB) mononuclear cells were first collected, and CD3+ T cells were separated. The cells were then transfected by lentivirus encoded with CD19 CD22 bispecific scFv sequences. The CAR-T cells produced in this way contained a 4-1BB co-stimulatory signal domain. The CAR-T cells were then cultured for 8-14 days until sufficient cells were harvested for infusion. All pts received conditioning regimen of fludarabine and cyclophosphamide intravenously for 3 consecutive days with doses of 30 mg/m2/day and 250 mg/m2/day, respectively before a single infusion of CAR-T cells. The level of infused CAR-T cell proliferation in PB was analyzed by qPCR and flow cytometry. The primary end points were to evaluate feasibility and toxicity, and the secondary end points included disease response and engraftment/persistence of infused CD19/CD22 dual CAR-T cells. Results From Feb. 2019 to 23 July. 2019, 17 patients (pts) with relapsed/refractory B-ALL including 4 pts who previously treated with CD19 CAR-T cells were enrolled and pts were treated with CD19/CD22 dual CAR-T GC022 (US NIH Clinical#: NCT03825731). Four were adults, 13 pediatrics (age 1-45, Table 1). The median bone marrow (BM) blasts was 19.09 (0.36-87.82) %. Four patients received a low-dose (2.5-5×105/kg) dual CAR-T, 7 received a medium-dose (1-2.5×106/kg) and 5, a high-dose (3-5×106/kg). One patient withdrew immediately before CAR-T infusion due to his personal issue. Anti-leukemic efficacy was evaluated in 11/16 pts (5 pts have not yet reached D15). The 3/4 pts received low dose of GC022 had no response to treatment and 1 had MRD-positive CR. Seven patients who received medium dose achieved 100% CR on D15, highlighting the dose-dependent anti-leukemic activity. Six out of seven pts had MRD negative CR in this medium dose group. Five pts in high dose group have not reached the time for evaluation. No one relapsed with a median observation time of 60 (7-139) days. Cellular kinetic data was analyzed. Median peak of CAR-T copies was 1.09 (0.0022-4.98) x105 copy number/µg PB genomic DNA (Fig.1). The proliferation of medium or high dose groups was significantly better than the low dose group 3.47(0.43-4.98) x105 vs. 0.023(0.0022-0.81) x105(P=0.02) and 2.02(1.89-2.16) x105 vs. 0.023(0.0022-0.81) x105(P=0.004), (Fig.2). The peaks of IL-6, IFN-γ, IL-10, and CD25 were observed around day 7-10. Sixteen out of seventeen pts had grade 0-1 cytokine release syndrome (CRS) and only 1 patient experienced grade 2 CRS. None developed neurotoxicity. Conclusion Our study demonstrates safety and technical feasibility of CD19 and CD22 dual CAR-T in treating patients with CD19+CD22+ relapsed/refractory B-ALL. A low toxicity with dose-dependent high CR rate including pts who previously treated with CD19 CAR-T cells were observed. Longer observation time and more patients are needed to evaluate a beneficial advantage of the CD19/CD22 dual CAR-T over CD19 CAR-T product. Disclosures No relevant conflicts of interest to declare.

Introduction CD19-targeting chimeric antigen receptor (CAR) T cell therapy has demonstrated high success; however, its therapeutic potential can still be further improved. In addition, the high cost and lengthy process of CAR-T production limit its broad application. We have developed a new platform termed FasT (F) CAR-T with shortened manufacturing time to one day (plus 7 days of additional testing for regulatory requirements). Here we report results from a pre-clinical study of FasT (F) CAR-T (GC007F) and a phase Ⅰ clinical trial to assess the safety and feasibility of treating patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Methods In this study, a second generation of CD19-directed CAR-T was manufactured using the FasT CAR-T platform. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis either from healthy donors for the pre-clinical study or from patients undergoing the clinical trial. T cells were separated and used for CAR-T generation. A xenograft mouse model was used to determine the efficacy of GC007F in vivo. Conventional (C) CAR-T derived from the same healthy donor were also made and tested in parallel for comparison. Between Feb. 2019 and July 2019, 10 adolescent and adult patients with CD19+ relapsed/refractory B-ALL were enrolled in a feasibility trial for CD19 FasT CAR-T (www.clinicaltrials.gov, NCT03825718). FasT CAR-T cells for all patients were successfully manufactured. All patients received a conditioning regimen of IV fludarabine (30mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single infusion of CAR-T cells. Six patients received a low-dose 6.5 (5.86-7.04) x104/kg of FasT CAR-T, 2 received a medium-dose 1 (1-1.16) x105/kg, and 1, a high-dose 1.56x105/kg. The primary end points of the study were to evaluate feasibility and toxicity, and the secondary end points included disease response and engraftment/persistence of infused FasT CAR-T cells. Results This preclinical study has demonstrated several significant improvements of CD19-directed F CAR-T over C CAR-T: 1) 5-30 fold superior expansion capability (p For the phase Ⅰ clinical trial, the median observation period was 86 days (37-166 days). The median percentage of pre-treatment bone marrow (BM) blasts was 9.05% (0.19-32.5%). On day 15 after CAR-T cell infusion, 10/10 (100%) cases achieved complete remission (CR) or CR with incomplete count recovery (CRi) and 9/10 (90%) had minimal residual disease (MRD)-negative CR. Four of ten patients had a good blood count recovery on day 15. The number further increased to 6/10 on day 30. Patient F15 had rapidly growing disease in that his PB blasts increased from 1% on enrollment to 7% immediately before CAR-T cells infusion, and increased to 77% on day 7 post infusion. Notwithstanding the rapid disease progression, the patient achieved MRD-positive CR on day 15 with residual 0.06% BM blasts. Five of ten patients were bridged into allogeneic hematopoietic stem cell transplantation (allo-HSCT). All 10 patients have remained in CR thus far. After CAR-T infusion, the level of infused CD19 FasT CAR-T cells in PB was analyzed by qPCR and flow cytometry. Superior in vivo proliferation and persistence were detected regardless of the infused CAR-T doses. The median peak level was reached on day 7 (7-10) with 2.1(0.22-5.2) x105 copy/µg PB genomic DNA (Fig. 2) and the median CAR-T expression ratio was 44.5 (13.6-69.5) %. The peaks of IL6, IFNγ, IL10, and CD25 were observed around day 7. Despite the achievement of a very high CR rate, 9/10 had grade 1 cytokine release syndrome (CRS) and only 1 patient experienced grade 3 CRS. None developed neurotoxicity. Conclusion This study has demonstrated that FasT CAR-T cells with superior expansion capability and younger/less exhausted phenotypes can be generated rapidly. This first-in-human clinical study showed that FasT CAR-T is safe and highly effective for treating patients with B-ALL. Disclosures No relevant conflicts of interest to declare.